The overall goal of this project is to elucidate the molecular basis for how protein acetyltransferases (PATs) recognize and acetylate their cognate protein substrates and how this process to modulated by protein cofactors and posttranslational modification. The most well studied protein acetyltransferases are the histone acetyltransferases (HATs) that acetylate histone, and in some cases other proteins, to mediate several different transcription-mediated biological processes including cell cycle progression, dosage compensation and hormone signaling; and aberrant HAT function has also been correlated with human disease, including leukemic translocations, solid tumors and metabolic disorders. HATs fall into at least four families based on sequence conservation, Gcn5/PCAF, MYST, p300/CBP and Rtt109. Over the last two funding periods, my laboratory reported on the structure and chemistry of these enzymes revealing that despite the sequence divergence between the HAT families, they contain a structurally related core region to mediate Ac-CoA cofactor and protein substrate binding but structurally divergent core flanking regions to mediated different modes of catalysis and other biological properties. Most recently, we have found that many HATs are regulated by autoacetylation and have identified HAT-selective inhibitors that may have therapeutic applications. More recent studies by other laboratories have also uncovered that over 2000 human proteins are acetylated, extending to many different types of proteins such as kinases and RNA processing factors and extending beyond nuclear processes such as vesicular trafficking and metabolism. These recent findings suggest that protein acetylation is just as important a posttranslational modification in signal transduction as protein phosphorylation and that acetyltransferases are just as important therapeutic targets as protein kinases. Despite our current understanding of HATs, several important questions about protein acetylation remain. These questions include, (1) How are PATs regulated by autoacetylation, (2) How are PATs regulated by associated cofactor proteins, (3) How do HATs differ from other PATs and (4) How do PATs recognize their cognate substrates. In this proposal we will study the Rtt109 HAT, the NATA N-amino protein acetyltransferase and the aTAT1/MEC-17 tubulin acetyltransferase as model systems to answer these mechanistic questions that will have important implications for understanding the biology of protein acetylation and for the development of potent and selective protein acetyltransferase inhibitors with possible therapeutic applications. The Specific Aims of the proposal are (1) Structure/Function studies of the Rtt109 histone acetyltransferase, (2) Structure/Function studies of the NATA N-amino protein acetyltransferase (NAT), and (3) Structure/Function of the aTAT1/MEC-17 a-tubulin acetyltransferase.